核仁蛋白RRP15在细胞有丝分裂期的表达与定位
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摘要
为了明确核糖体RNA加工蛋白15(RRP15)在细胞周期不同时期的表达情况与亚细胞定位,以稳定表达GFP-RRP15的He La细胞为实验材料,利用细胞同步化以及蛋白免疫印迹方法研究RRP15在细胞周期不同时期的表达情况,利用细胞免疫荧光染色以及活细胞成像检测RRP15在有丝分裂期的亚细胞定位,利用染色体提取探究RRP15与有丝分裂期细胞染色体的关系.结果发现,RRP15在整个细胞周期中均有稳定表达,且在G1期表达微量上调;在有丝分裂期,RRP15定位于染色体外周和中小体,始终伴随染色体,且染色体外周定位不依赖于DNA.
To explore the expression and localization of ribosome RNA processing protein 15(RRP15)in mitosis,He La cells which can stably express GFP-RRP15 were adopted as the experimental materials,and the expression of RRP15 in different periods of cell cycle was determined by cell synchronization and Western blot. The subcellular localization of RRP15 in mitosis was detected by using immunofluorescence staining and living cell imaging. The relationship between RRP15 and mitotic cell chromosome was studied by using chromosome spreading assay. The results showed that RRP15 was expressed throughout the cell cycle,and the expression was subtly increased in G1 phase. Furthermore,RRP15 was localized on perichromosomal layer in mitosis and midbody in telophase. In addition,the localization of RRP15 on perichromosomal layer was independent on chromosome DNA.
To explore the expression and localization of ribosome RNA processing protein 15(RRP15)in mitosis,He La cells which can stably express GFP-RRP15 were adopted as the experimental materials,and the expression of RRP15 in different periods of cell cycle was determined by cell synchronization and Western blot. The subcellular localization of RRP15 in mitosis was detected by using immunofluorescence staining and living cell imaging. The relationship between RRP15 and mitotic cell chromosome was studied by using chromosome spreading assay. The results showed that RRP15 was expressed throughout the cell cycle,and the expression was subtly increased in G1 phase. Furthermore,RRP15 was localized on perichromosomal layer in mitosis and midbody in telophase. In addition,the localization of RRP15 on perichromosomal layer was independent on chromosome DNA.
引文
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[3]ZHU Y C,LI B,WU T H,et al.Cell cycle and histone modification genes were decreased in placenta tissue from unexplained early miscarriage[J].Gene,2017,636(1):17-22.
[4]BARRETT A K,RUBIN S M.He avy metal in cancer:The cell cycle jams with arsenic[J].Cell Cycle,2017,16(18):1641-1642.
[5]KWASNIK A,VON KRIEGSHEIM A,IRVING A,et al.Potential mechanisms of calcium dependent regulation of the mammalian cell cycle revealed by comprehensive unbiased label-free n LC-MS/MS quantitative proteomics[J].J Proteomics,2018,170(1):151-166.
[6]MATSUDA S,HAMMAKER D,TOPOLEWSKI K,et al.Regulation of the cell cycle and inflammatory arthritis by the transcription cofactor LBH gene[J].J Immunol,2017,199(7):2316-2322.
[7]WANG M,LEMOS B.Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context,nucleolus activity,and proliferation[J].PLo S Genetics,2017,13(9):e1006994.
[8]ANTONIALI G,LIRUSSI L,POLETTO M,et al.Emerging roles of the nucleolus in regulating the DNA damage response:The noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example[J].Antioxid Redox Signal,2014,20(4):621-639.
[9]CMARKO D,SMIGOVA J,MINICHOVA L,et al.Nucleolus:The ribosome factory[J].Histol Histopathol,2008,23(10):1291-1298.
[10]FULKA H,AOKI F.Nucleolus precursor bodies and ribosome biogenesis in early mammalian embryos:Old theories and new discoveries[J].Biol Reprod,2016,94(6):143.
[11]MACCALLUM D E,HALL P A.The location of p Ki67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle[J].J Pathol,2000,190(5):537-544.
[12]JIANG Z,YANG J,DAI A,et al.Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress[J].BMC Genomics,2017,18(1):638.
[13]SLOAN K E,BOHNSACK M T,WATKINS N J.The 5S RNP couples p53 homeostasis to ribosome biogenesis and nucleolar stress[J].Cell Rep,2013,5(1):237-247.
[14]FUMAGALLI S,DI CARA A,NEB-GULATI A,et al.Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rp L11-translation-dependent mechanism of p53 induction[J].Nat Cell Biol,2009,11(4):501-508.
[15]MICHAEL D,OREN M.The p53-Mdm2 module and the ubiquitin system[J].Semin Cancer Biol,2003,13(1):49-58.
[16]ZHANG Y P,WOLF G W,BHAT K,et al.Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway[J].Mol Cell Biol,2003,23(23):8902-8912.
[17]DONG Z X,ZHU C J,ZHAN Q,et al.The roles of RRP15 in nucleolar formation,ribosome biogenesis and checkpoint control in human cells[J].Oncotarget,2017,8(8):13240-1352.
[18]DE MARCHIS M L,GIORGI A,SCHININA M E,et al.Rrp15p,a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation[J].RNA,2005,11(4):495-502.
[19]WU T,REN M X,CHEN G P,et al.Rrp15 affects cell cycle,proliferation,and apoptosis in NIH3T3 cells[J].FEBS Open Bio,2016,6(11):1085-1092.
[20]PRASANNA P G,ESCALADA N D,BLAKELY W F.Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes:A simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry[J].Mutat Res,2000,466(2):131-141.
[21]KUHN R,PRAVTCHEVA D,RUDDLE F,et al.The gene encoding peripheral myelin protein zero is located on mouse chromosome 1[J].J Neurosci,1990,10(1):205-209.